Mucins are large ( greater than 200 kDa) glycoproteins with a high carbohydrate content (50-90% by weight) expressed by a variety of normal and malignant epithelial cells (Strous el al., Crit. Rev. Biochem. Mol. Biol. 27:57 (1992); Devine et al., BioEssays 14:619 (1992)). Among the human mucins, MUC-1 is unique in its cell surface transmembrane expression (Gendler et al., J. Biol. Chem. 265:15286 (1990); Siddiqui et al. Proc. Natl. Acad. Sci. USA 85:2320 (1988); Gendler et al., Proc. Natl. Acad. Sci. USA 84:6060 (1987); Ligtenberg et al., J. Biol. Chem. 265:5573 (1990)).
MUC-1 mucin contains a polypeptide core consisting of 30-100 repeats of a 20 amino acid sequence (Gendler et al, J. Biol. Chem. 265:15286 (1990). The presence of large amounts of oligosaccharides attached along the length of the polypeptide core of MUC-1 mucin enhances its rigidity, resulting in large flexible rod-like molecules that may extend several hundred nanometers from the apical epithelial cell surface into the lumens of ducts and glands (Bramwell et at, J. Cell Sci. 86:249 (1986)).
Adenocarcinoma patients with elevated serum MUC-1 mucin levels have higher numbers of T-cells expressing CD69, an early activation marker, than the patients with normal serum MUC-1 levels (Reddish et al., Cancer Immunol. Immunother. 42:303 (1996); Bowen-Yacyshyn et al., Int. J. Cancer 61:470 (1995). It was hypothesized that patients with high serum MUC-1 levels and high numbers of CD6930  peripheral blood T-lymphocytes were in a state of T-cell anergy (Reddish et al., Cancer Immunol. Immunother. 42:303 (1996)) similar to tumor infiltrating lymphocytes (TILs), which are CD69+ but appear to be xe2x80x9cfrozenxe2x80x9d in an early activation state and unable to express normal interleukin-2 (IL-2) and L-2R levels (Alexander et al., J. Immunother. 17:39 (1995); Berd et al., Cancer Immunol. Immunother. 39:141 (1994); Barnd et al., Proc. Natl. Acad. Sci. USA 86:7159 (1989).
Elevated levels of serum MUC-1 are associated with poor survival and a lower anti-cancer immune response of metastatic breast, colorectal and ovarian cancer patients following immunotherapy (Bowen-Yacyshyn et al., 1995 Int. J. Cancer 61:470, MacLean et al., J.Immunother. 20:70 (1997)). Cumulatively, all of these results are consistent with an immunosuppressive role for MUC-1 mucin.
Direct demonstration of an immunosuppressive role of cancer associated MUC-1 mucin came from recent work (Agrawal et al., Nature Med. 4:43 (1998)) showing that cancer associated, affinity purified, MUC-1 mucin and synthetic tandem repeats of MUC-1 polypeptide core inhibited human T-cell proliferative responses to polyclonal stimuli. The degree of inhibition of T-cell proliferation was directly proportional to the number of tandem repeats present on MUC-1 polypeptide core synthetic peptides.
This inhibition was reversible by adding a 16 mer ( less than 1 tandem repeat of the polypeptide core) MUC-1 synthetic peptide (Agrawal et al., Nature Med. 4:43), which confirms the role of the MUC-1 polypeptide core in the inhibition of T-cell responses and suggests an inhibitory mechanism, which involves cross-linking of a T-cell surface molecule. The observation that addition of exogenous IL-2 or anti-CD28 monoclonal antibody (mAb) reversed the cancer associated MUC-1 mucin induced inhibition of T-cell response is consistent with the mechanism of inhibition being anergy (Agrawal et al., Nature Med. 4:43). Our understanding of the immunoregulatory role of cancer associated MUC-1 mucin has revealed some of the intricate mechanisms tumor cells use to regulate immune responses for their enhanced survival.
Aside from direct immmunomodulatory functions, other functions have been proposed for MUC-1 mucin (Gendler et al., Ann. Rev. Physiol. 57:607(1995)) which involve steric hindrance by the large glycosylated extracellular domain of cell-cell or cell-substratum interactions, remodeling the cytoskeletal network, or by down-regulating the activities of other molecules such as catenins, cadherins or Integrins via signal transduction events (Yamamoto et al., J. Biol. Chem. 272:12492 (1997); Parry et al., Exp. Cell Res. 188:302 (1990). Its cytoplasmic tail is phosphorylated consistent with a transmembrane signal transduction function for MUC-1 (Pandey et al., Cancer Res. 55:40003 (1995); Zrihan-Licht et al., FEBS Lett. 356:130 (1994); Mockensturm-Gardner et al., Mol. Biol. Cell 7:434a (1996); Mockensturm-Gardner et al, Proc. Amer. Assn. Cancer Res. 39:375a (1998).
Paradoxically, in previous studies MUC-1 mucin has been proposed to act both as an anti-adhesive as well as an adhesive molecule. The extended conformation of the extracellular domain of MUC-1 mucin may contribute to the anti-adhesive properties, resulting in reduced cell-cell aggregation and decreased adherence to extracellular matrix components in in vitro adhesion assays (Ligtenberg et al., 1992 Cancer Res. 52:2318; Wesseling et al., 1995 J. Cell Biol. 129:255; Wesseling et al., 1996 Mol. Biol. Cell 7:565). Thus, MUC-1 mucin may protect cancer cells from destruction by natural killer or other immune cells (Hayes et al., 1990 J. Immunol. 145:962, Ogata et al, 1992 Cancer Res. 52:4741, Zhang et al., 1997 Cell. Immunol. 66:158; van de Wiel-van Kemenade et al., 1993 J. Immunol. 151:767).
MUC-1 on cancer cells can also have adhesive features, as it expresses carbohydrate structures that may be ligands for selectin-like molecules on endothelial cells (Baeckstrom et al., 1991 J. Biol. Chem. 266:21537; Hanski et al., 1993 Cancer Res. 53:4082; Sikut et al., 1996 Int. J. Cancer 66:617; Zhang et al., 1997 Tumor Biol. 18:175; Zhang et al., 1996 J. Cell. Biochem. 60:538). MUC-1 mucin has also been shown to be a ligand for ICAM-1 (Regimbald et al., 1996 Cancer Res. 56:4244), another adhesion molecule involved in cell-cell interactions. MUC-1 can be shed from tumors and detected in serum (Hayes et al., 1985 J. Clin. Invest. 75:1671; Burchell et al., 1984 Int. J. Cancer 34:763; Boshell et al., 1992 Biochem Biophys. Res. Commun. 185:1; Williams et al., 1990 Biochem. Biophys. Res. Commun. 170:1331). The presence of soluble MUC-1 has been shown to inhibit adhesive interactions of migrating cells with endothelial cells (Zhang et al., 1997 Tumor Biol. 18:175) and thus could cause decreased recruitment of inflammatory cells to the tumor site.
Although it has primarily been studied based on its association with cancer, MUC-1 is in fact expressed by a variety of normal tissues. A number of secretory epithelial cells, for example, express and secrete MUC-1 mucin. Although, this MUC-1 is highly glycosylated, and is therefore somewhat different than cancer-associated MUC-1, which is under glycosylated.
Various glycoforms of MUC-1 mucin (similar to those of cancer associated MUC-1 mucin) have been found to be present in endometrium and in the serum of pregnant women. McGuckin et al., Tumour Biol. 15:33 (1994). During the menstrual cycle, the abundance of MUC-1 varies in human endometrium. Moreover, progesterone up regulates the transcription of MUC-1 and maximum MUC-1 expression appears in the implantation phase. Hey et al., J. Clin. Endocrinol. Metab. 78:337 (1994).
Interestingly, it has been shown that high levels of progesterone present during days 14-28 of the menstrual cycle are associated with inhibition of CTL activity in the uterus. Consequently, the down-regulation of CTL activity may allow implantation of a semi-allogeneic embryo, which would be otherwise be rejected. White et al., J. Immunol. 158:3017 (1997). The mechanism of this T-cell down-regulation, however, is unknown. Indeed, the art is generally deficient in its knowledge regarding T-cell activation and de-activation.
T-cell activation is an indicator of the immune state and thus is useful in monitoring a variety of diseases. For example, certain autoimmune diseases are etiologically linked to T-cell activation. Moreover, the ability to control the state of T-cell activation would, likewise, be useful in treating a wide variety of disorders.
A need exists, therefore, in the art for the elucidation of a fundamental pathway involved in the regulation of T-cell activation. Provided such a pathway, certain diagnostic and medicinal agents will be made available to the art. The present invention, as detailed below, describes such a novel fundamental pathway as well as a variety of compounds for modulating that pathway, which have certain diagnostic and therapeutic applications.
It is therefore, one object of the invention to provide reagents and methods for determining the state of T-cell activation. According to this aspect of the invention, methods are provided which comprise detecting the amount of MUC-1 expression in a T-cell-containing sample and comparing said amount to a non-activated T-cell-containing control. In one embodiment, detection involves quantifying the amount of MUC-1 expression, preferably by contacting the sample with an antibody which is specific for MUC-1. In another embodiment detecting can be accomplished by contacting the sample with a polynucleotide probe which is capable of specifically detecting a MUC-1-associated nucleotide sequence. In yet another embodiment, methods of ascertaining global immune activation are provided. In a typical method, a sample is provided from a patient and the amount of MUC-1 present in the sample is detected.
It is another object of the invention to provide methods for modulating T-cell activation, generally in the context of therapeutic methods. According to this object, methods are provided which entail treatment with compounds that either promote or antagonize the activity of MUC-1, thus altering the balance of T-cell activation in patients in need of such treatment.